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Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells.

ABSTRACT: In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

SUBMITTER: Fujihara C

PROVIDER: S-EPMC2934693 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells.

Fujihara Chiharu C Yamada Satoru S Ozaki Nobuhiro N Takeshita Nobuo N Kawaki Harumi H Takano-Yamamoto Teruko T Murakami Shinya S

The Journal of biological chemistry 20100624 36

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glu ...[more]

PMID: 20576613

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Project description:The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

Project description:BACKGROUND:The oral cavity is a complex environment in which periodontal tissue is constantly stimulated by external microorganisms and mechanical forces. Proper mechanical force helps maintain periodontal tissue homeostasis, and improper inflammatory response can break the balance. Periodontal ligament (PDL) cells play crucial roles in responding to these challenges and maintaining the homeostasis of periodontal tissue. However, the mechanisms underlying PDL cell property changes induced by inflammatory and mechanical force microenvironments are still unclear. Recent studies have shown that exosomes function as a means of cell-cell and cell-matrix communication in biological processes. METHODS:Human periodontal ligament stem cells (HPDLSCs) were tested by the CCK8 assay, EdU, alizarin red, and ALP staining to evaluate the functions of exosomes induced by a mechanical strain. MicroRNA sequencing was used to find the discrepancy miRNA in exosomes. In addition, real-time PCR, FISH, luciferase reporter assay, and western blotting assay were used to investigate the mechanism of miR-181b-5p regulating proliferation and osteogenic differentiation through the PTEN/AKT pathway. RESULTS:In this study, the exosomes secreted by MLO-Y4 cells exposed to mechanical strain (Exosome-MS) contributed to HPDLSC proliferation and osteogenic differentiation. High-throughput miRNA sequencing showed that miR181b-5p was upregulated in Exosome-MS compared to the exosomes derived from MLO-Y4 cells lacking mechanical strain. The luciferase reporter assay demonstrated that miR-181b-5p may target phosphatase tension homolog deletion (PTEN). In addition, PTEN was negatively regulated by overexpressing miR-181b-5p. Real-time PCR and western blotting assay verified that miR-181b-5p enhanced the protein kinase B (PKB, also known as AKT) activity and improved downstream factor transcription. Furthermore, miR-181b-5p effectively ameliorated the inhibition of HPDLSC proliferation and promoted HPDLSC induced by inflammation. CONCLUSIONS:This study concluded that exosomes induced by mechanical strain promote HPDLSC proliferation via the miR-181b-5p/PTEN/AKT signaling pathway and promote HPDLSC osteogenic differentiation by BMP2/Runx2, suggesting a potential mechanism for maintaining periodontal homeostasis.

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Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells.

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Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells.

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